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Angiogenesis is required for normal follicular development but the role of gonadotrophins in the control of follicular angiogenesis remains to be elucidated. This study investigated the effects of treatment with GnRH antagonist in vivo on follicular development and angiogenesis in the marmoset. GnRH antagonist was administered on either follicular day 0 or day 5 of the 10-day follicular phase with ovaries collected on day 10. Ovaries from control marmosets were studied at day 5 (mid follicular phase) and day 10 (periovulatory period). Ovaries were fixed, serial sectioned and subjected to morphological analysis and immunocytochemistry to determine cell proliferation and follicular endothelial cell area and in situ hybridization to assess changes in expression of vascular endothelial growth factor (VEGF). Treatment with GnRH antagonist from day 0-10 resulted in an absence of dominant preovulatory follicles seen in controls. In the remaining tertiary follicles granulosa, theca and endothelial cell proliferation was reduced, resulting in a minor reduction in vascular density. However, VEGF mRNA expression was unaffected by treatment. Treatment from day 5-10 did not prevent development of ovulatory size follicles, but they were atretic and lacked VEGF mRNA. These results suggest that while VEGF expression in the preovulatory follicle is under gonadotrophic control it is not dependent on normal gonadotrophin secretion in tertiary follicles, indicating that there are other paracrine factors regulating VEGF expression in the developing ovarian follicle.

Problem: The effect of neonatal gonadotropin releasing hormone (GnRH) antagonist (Ant) treatment and seasonality on immune system development and function was investigated in male primates., Method of Study: Neonatal male rhesus monkeys and marmosets were treated with Ant, and its effect on immune system morphology, circulating lymphocyte subsets, and cell- and humorally-mediated immune responses was assessed during development. In adult rhesus monkeys, we correlated seasonal changes in immune function with circannual fluctuations in immunoactive hormones., Results: In neonatal marmosets, Ant reduced the number of B cells and T cells in the thymic medulla and T cells in the periarterial lymphatic sheaths (PALS) of the spleen. Ant also altered the development of, but did not permanently impair, the proliferative index (PI) of blood lymphocytes to mitogens. In vitro treatment of control lymphocytes with GnRH analogues altered their response to these proliferative agents. In neonatal rhesus monkeys, Ant treatment increased the frequency of clinical problems, lowered circulating levels of lymphocytes, total T cells, CD8+ T cells and B cells, and altered the PI of lymphocytes to mitogens. As adults, the cell- and humorally-mediated immune responses remained impaired. We also documented seasonal fluctuations in the prevalence of diseases, circulating immune cells and immune function in rhesus monkeys. The number of cases of campylobacteriosis and shigellosis was lowest in the winter and highest in the spring. Circulating numbers of white blood cells (WBC) and neutrophils and the PI of lymphocytes to mitogens were higher in the winter than in the summer. Natural killer cell activity also varied with season. Cortisol and leptin secretion exhibited circannual rhythms, rising in concert with decreasing photoperiod and increasing testicular activity in the fall. Conversely, prolactin levels declined with decreasing photoperiod and then rose in the spring., Conclusion: Neonatal exposure of male primates to Ant appears to alter early postnatal programming of immune function. In the rhesus monkey, immune function shows seasonal fluctuations that may be driven by circannual changes in the secretion of immunoactive hormones.

The primary purpose of this study was to establish whether Sertoli cells proliferate in the neonatal period in the marmoset monkey (Callithrix jacchus) and whether administration of a long-acting GnRH antagonist (GnRHa) during this phase induced any transient or permanent effects on Sertoli cell number or on any other aspect of testicular development. Male marmoset co-twins (n = 9) were treated during Weeks 1-14 with either vehicle or GnRHa. Four sets of co-twins were examined at Weeks 18-22 (start of infancy) and 5 sets in adulthood (92+ wk), and Sertoli cell number was determined using either the nucleator or optical disector methods; other testicular morphometric analyses (e.g., germ cell volume, Leydig cell volume) used standard point-counting. Data for the marmoset were compared with that obtained in similarly treated rats. Sertoli cell number in marmosets treated neonatally with GnRHa was reduced by 35% compared with that of controls at Weeks 18-22 but was comparable to control values in adulthood. However, seminiferous epithelium volume was reduced significantly in adult marmosets treated neonatally with GnRHa, and there was a tendency for reduced germ cell volume per Sertoli cell. In the same animals, there was significant expansion of the interstitium and an increase in Leydig cell volume per testis when compared with co-twin controls; a similar increase in Leydig cell volume was evident in adult rats treated neonatally with GnRHa. Comparison of Sertoli cell numbers in 6 infantile (18-24 wk) and 10 adult marmosets showed that adult numbers of Sertoli cells were present by the start of infancy but, unlike rats, marmosets were still able to replicate Sertoli cells beyond this period. However, marmoset Sertoli cells supported only approximately 20% of the germ cell volume supported by rat Sertoli cells, indicative of poor efficiency of spermatogenesis, as shown previously in the human. This finding, together with the demonstration of a temporal pattern of Sertoli cell replication similar to that in the human, supports the use of marmosets as a model for human male testicular development and function.

Morphological changes in the corpus luteum following natural and induced luteolysis in the marmoset were investigated by light and electron microscopy. Functional corpora lutea were studied in the mid and late luteal phase, naturally regressed corpora lutea in the early and late follicular phase, and corpora lutea induced to regress by administration of GnRH antagonist or prostaglandin F(2alpha) analogue in the midluteal phase. Natural luteolysis was associated with lutein cell atrophy, condensation of cytoplasmic inclusions and organelles, and accumulation of lipid. GnRH antagonist treatment resulted in aggregations of smooth membranes and myelin-like bodies in the cytoplasm of the lutein cells together with complex aggregations of degenerative cells. After prostaglandin treatment, the lutein cells contained numerous small and large vesicles; as the degenerative changes advanced, these vesicles coalesced into alveolar-type vacuoles, and nuclei involuted. These results show that in the marmoset, natural luteolysis and the two luteolytic treatments reveal different forms of luteal degeneration and cell death, none of which fit the ultrastructural criteria for apoptosis. More emphasis needs to be placed on understanding these predominant nonapoptotic forms of cell death in order to elucidate the process of luteolysis in the primate.

Problem: We examined the effect of neonatal treatment with a gonadotropin-releasing hormone (GnRH) antagonist (antide) on the development of cell-mediated immunity in male marmosets., Method of Study: Neonatal marmoset twins were treated with either vehicle or antide, and the proliferative response (PR) of lymphoid tissue to mitogens was assessed during infancy, the peripubertal period, and adulthood., Results: Basal proliferation of peripheral blood mononuclear cells (PBMC) from treated peripubertal twins was elevated above control values, but the PR of the cells to T and B cell mitogens was subnormal. Conversely, PBMC from treated infants exhibited an enhanced PR to some of the mitogens employed. In vitro culturing of thymocytes (control or treated) from the three developmental stages with either antide or a GnRH agonist increased basal proliferation, but decreased the PR to mitogens by 60-80%., Conclusion: Neonatal treatment with antide alters development of, but does not permanently impair, cell-mediated immunity in the marmoset. GnRH appears to modulate immune responses throughout development in the primate.

Little is known of the cell biology of Leydig cells during the neonatal activation of the hypothalamic-pituitary-testicular (HPT) axis. The current study examined the effect of blockade of the HPT axis with a GnRH antagonist (antide) on the neonatal population of Leydig cells in the new world primate, the common marmoset. Three sets of twins, age 7 weeks, were studied: in each pair one twin was used as a control, while the other received treatment with GnRH antagonist from the day of birth to suppress pituitary gonadotrophin secretion. Leydig cells of treated animals were dramatically different from those of controls. The cells were atrophic and exhibited very irregular nuclei. The organelles involved in steroid synthesis were reduced to the extent to being barely evident. The smooth endoplasmic reticulum (SER) was greatly diminished in quantity and distribution. The usual form of the SER (anastomosing tubules) was not evident, but, instead, the SER was relatively unbranched. Peroxisomes, organelles involved in transfer of cholesterol to the mitochondria, were greatly reduced in number. Mitochondria were relatively sparse and exhibited a non-typical morphology, as tubular elements of the cristae were rarely evident. Thus, the central apparatus in steroid production, the SER, mitochondria and peroxisomes, was essentially shut down in the GnRH-antagonist-treated animals. Storage of cholesterol, the precursor of steroid biosynthesis, was also not in evidence, as lipid droplets were extremely rare. Two prominent features of control in neonatal marmoset Leydig cells, the membranofibrillar inclusion (MFI) and basal laminae, remain prominent in the Leydig cells of treated animals. Evidence of apoptosis was not observed. These results provide strong support that the gonadotrophic hormones are the primary regulator of neonatal Leydig cell development in primates, and also suggest cell regression, rather than apoptosis, being the mechanism of this inhibition.

The hypothalamo-pituitary-gonadal axis matures during fetal life and may be susceptible to adverse effects. Model systems can be used to understand its physiological role. The aim of this study was to determine whether antibodies to GnRH, administered to the mother, can cross the ovine fetal-placental barrier and suppress fetal gonadotrophin secretion. Maternal passive immunization to GnRH in pregnant Greyface ewes (day 103 of gestation) resulted in GnRH antibody titres of from 1:6000 to 1:9000 after 1 day and a suppression of the pulsatile secretion of fetal LH after 2 days. Fetal FSH concentrations declined gradually over the 11 days of the experiment and were only significantly different from control animals immunized against BSA in male fetuses. The slower decrease in fetal FSH concentrations than in LH concentrations shows that the secretion of FSH, unlike that of LH, is not dependent on short term changes in GnRH release. The lack of a suppressive effect of the maternal GnRH immunoneutralization on female fetal FSH secretion may be due to removal of the negative feedback effect of oestradiol and, possibly, inhibin. There was no sexual dimorphism in the effect of maternal GnRH immunoneutralization on fetal GnRH antibody titres or fetal LH secretion. These findings show that maternal passive immunization against GnRH results in GnRH antibodies crossing the fetal-placental barrier and suppressing fetal LH and FSH secretion in males, but only suppressing LH secretion in the females. Although the lack of effect on FSH secretion in the females needs to be investigated further, the present study provides evidence of a non-invasive procedure for blocking fetal gonadotrophin secretion which may be used to investigate hypothalamo-pituitary-gonadal function during early gestation in sheep.

Problem: The effect of neonatal treatment with a gonadotropin releasing hormone (GnRH) antagonist on the morphology and distribution of lymphocytes in lymphoid tissue of the infant marmoset was examined., Method of Study: From a screened panel of antihuman antibodies for specific immune cells, antibodies for the CD20 and CD3 antigens showed excellent reactivity with marmoset tissue. Five sets of marmoset twins were treated with either the GnRH antagonist or a vehicle from birth, and were euthanized at 7 to 9 (3 sets) or 16 to 20 weeks (2 sets) of age. The spleen, thymus, and inguinal lymph nodes from each animal were processed for immunocytochemistry, and the number of cells expressing the CD20 and CD3 antigens were quantified., Results: Control twins exhibited high plasma levels of testosterone, characteristic of the neonatal period, whereas testosterone concentrations were reduced (P = 0.001) to detection limits in the GnRH antagonist-treated twins. Microscopic evaluation suggested that treatment reduced the volume and cellularity of the thymic cortex, resulting in a decrease in the cortical-to-medullary ratio. Treatment reduced (P = 0.046) the number of thymocytes expressing the B-cell antigen (CD20) and marginally lowered (P = 0.067) the number expressing the T-cell antigen (CD3) in the thymic medulla. In the spleens of treated animals, periarterial lymphatic sheaths were less prominent on microscopic examination, and there were marginally fewer (P = 0.064) CD3+ cells. Numbers of CD20+ lymphocytes in the peripheral white pulp of the spleen and in the germinal centers of the lymph nodes, or CD3+ cells in the paracortex and germinal centers of the lymph nodes, were not altered by treatment., Conclusion: Neonatal treatment with a GnRH antagonist may alter maturational processes for B and T cells in the thymus and spleen of the marmoset and may deprive the immune system of its normal sensitivity to GnRH at a potentially critical time in development.

Male marmoset monkeys which had received gonadotrophin-releasing hormone (GnRH) antagonist treatment as neonates to block the postnatal increase in testosterone were studied, with the object of determining potential long-term effects of treatment on the reproductive system, including tests of fertilising capacity. To obtain information on the nature of the circulating testosterone during this neonatal period, sequential blood samples were collected from a further control group of ten neonates, aged between birth and 3 months, and from 11 adult, normally fertile males, to examine the relative proportions of free, sex-hormone-binding globulin (SHBG)-bound, and non-SHBG-bound testosterone. In control neonates, 11% of the circulating testosterone was free, and a further 19% non-SHBG-bound, and therefore presumed to be biologically available. The remaining 70% was SHBG-bound and considered to be biologically inert. This indicates that the neonatal increase in marmoset testosterone has a biological function. After pairing with females, time to first positive vaginal lavage and first delivery was similar for females, whether they were with control or treated male partners. Pregnancy outcome, in terms of number of young delivered and sex ratio, did not differ. This indicates that there appear to be no long-term sequelae in terms of procreative ability in male marmosets treated neonatally with a GnRH antagonist. Autopsy revealed no gross changes, except in the thymus, which was significantly heavier in the treated group. These results indicate that, although the circulating testosterone is in a biologically active form during the neonatal period, inhibition of testicular function in the neonate is without major effect on the adult male reproductive system. Treatment with a GnRH antagonist may have long-term effects on the immune system.

We examined the effect of reversibly suppressing pituitary-testicular function during the neonatal period on developmental changes in inhibin-B and FSH secretion in male rhesus monkeys. Infants were treated with either vehicle, a GnRH antagonist (Ant) or the Ant and androgen (Ant/And) for the first 4 postnatal months, and the effects on serum inhibin-B and FSH were monitored during the neonatal and peripubertal periods. In neonates, Ant or Ant/And treatment lowered both serum FSH and inhibin-B levels. By 12 months of age, inhibin-B concentrations no longer differed across treatment groups. A major increase in inhibin-B occurred between 27-36 months of age (late prepubertal period) in all groups, but levels were lower at 33 and 36 months of age in Ant/And-treated animals than in controls. These differences most likely were related to fewer Ant/And-treated animals achieving sexual maturity during their fourth year of life. Regardless of treatment, inhibin-B levels were higher in those that were destined to become mature (in year 4) than in those that were not. During the late prepubertal period, serum inhibin-B was positively correlated with age and testicular volume, but not with serum LH or testosterone. After this period (39-52 months of age), inhibin-B no longer correlated with these parameters. FSH levels were near or below detection limits in most peripubertal animals, but FSH was detectable in fewer samples from control than treated animals. The data suggest that inhibin-B secretion in the neonate is driven by gonadotropin secretion, but during the juvenile hiatus in gonadotropin secretion, the monkey testis continues to produce substantial amounts of this hormone.

The determination of the efficacy of gonadotrophin-releasing hormone (GnRH) antagonists in blocking the luteinizing hormone (LH) surge and luteal function is important for our understanding of the control of the menstrual cycle and for clinical application. GnRH antagonists have failed to block the LH surge reliably in the non-human primate. The aim of the study was to utilize high dose GnRH antagonist treatment administered during the late follicular phase of the menstrual cycle to block the pre-ovulatory LH surge. It was postulated that the LH surge would be prevented in all animals, but if this failed subsequent luteal function would be blocked by continued suppression of LH, since the early corpus luteum is susceptible to inhibition by GnRH antagonist treatment. A group of 16 adult female stumptailed macaques (Macaca arctoides) with regular menstrual cycles were selected. The GnRH antagonist [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,D-(Hci)6, Lys(iPr)8,D-Ala10]GnRH (Antarelix) (concentration 10 mg/ml) was administered as three daily s.c. injections, at a dose of 1 mg/kg on days 11, 12 and 13 of the follicular phase of the menstrual cycle. Of nine macaques in which it was judged that the treatment was commenced within 1 day of the expected LH surge (serum oestradiol >400 pmol/l), six demonstrated a decline in serum oestradiol concentrations, a total block of the LH/follicle stimulating hormone (FSH) surge and inhibition of ovulation as judged by an absence of a rise in progesterone concentrations. In the three other animals in this category, a partial LH surge occurred, but this failed to result in a functional corpus luteum. In a further three animals treatment was initiated on the day of the LH surge, and again there was absence of a subsequently functional corpus luteum. These results show that GnRH is involved at the time of the mid-cycle LH/FSH surge in the non-human primate. Initiation of high dose GnRH antagonist treatment during the periovulatory period abolishes luteal function irrespective of its effects upon the LH surge because of its long-term action and resultant withdrawal of luteal support.

In primates, plasma testosterone concentrations are elevated for some 3 months from birth. The function of this rise is uncertain, but studies in rats suggest that its prevention by castration or administration of gonadotrophin hormone-releasing hormone (GnRH) analogues has effects on development and expression of social and sexual behaviours, and adverse long-term effects on fertility. The consequences of suppression of this rise in testosterone by treatment with the GnRH antagonist antide have been investigated in the marmoset monkey. Eight sets of male:male twins were used, one of each set receiving s.c. injections of antide (10 mg/kg), on days 0, 3 and 7, then weekly from birth to 98 days of age, with the twin receiving vehicle only. Plasma samples were taken at weekly intervals for the determination of testosterone concentrations from birth until 2 years of age. Treatment with antide completely abolished the neonatal rise in testosterone seen in control animals. The timing of the onset of the pubertal testosterone rise was not significantly affected by treatment; however, the subsequent pattern of circulating testosterone showed a tendency to decreased plasma concentrations in the neonatally treated group from weeks 25 to 42, relative to controls, and this difference was significant between 43 and 70 weeks. This was associated with a similar depression in bioactive LH concentrations around this time. Thereafter, the testosterone concentrations were similar between treated and control groups. There was no effect of treatment on growth, based on sequential body weight data. At 20 months the animals underwent behaviour tests with ovariectomized females.(ABSTRACT TRUNCATED AT 250 WORDS)

GnRH antagonists have potential for use in postpartum contraception, as it is likely that they would be effective in maintaining the inhibition of ovulation associated with lactation, but possible effects on the infant by transfer of antagonist via breast milk are unknown. The aim of this study was to establish whether chronic treatment with a GnRH antagonist would prevent ovulation throughout the period of lactation using the marmoset monkey as a model and to evaluate the effects of the presence of GnRH antagonist in breast milk on the postnatal rise in plasma testosterone concentrations in male infants. Mothers who had delivered male twins were selected. Starting within 3 days postpartum, mothers (n = 5/group) were treated with either 6.0 or 0.6 mg/kg of the GnRH antagonist antide, sc, once per week for 11 weeks. Ten postpartum lactating females acted as controls. All animals were housed in family groups with fertile males. To determine possible effects on the postnatal rise in testosterone, plasma samples were collected from the male infants of the high dose antide-treated mothers at weekly intervals for 12 weeks. Plasma progesterone concentrations were monitored in lactating mothers until establishment of pregnancy in both treated and control groups. Concentrations of antide were determined by RIA in plasma from mothers in the high dose group, in their milk, and in pooled plasma samples from their infants. Ovulation followed by pregnancy occurred in all of the control animals. No ovulations occurred during treatment with antide. Antide was present throughout treatment in plasma and breast milk in the mother. In male infants feeding from mothers receiving high dose treatment, antide was not detectable in infant plasma, and plasma testosterone concentrations were within the normal range for male neonates in our colony. These results indicate that the GnRH antagonist has potential as a method of contraception postpartum without affecting the postnatal rise in testosterone in male infants.

The production of chemical analogues of GnRH permits direct suppression of the pituitary-gonadal axis at the level of the gonadotroph. Continuous administration of GnRH agonists desensitizes the gonadotroph and ovulation is uniformly prevented, forming the practical basis for use in contraception. However, long-term treatment is constrained by variable effects on oestrogen secretion, which cause irregular bleeding patterns on the one hand and risks of hypo-oestrogenism on the other. Their use as a post-partum contraceptive has attractions because any analogue in milk should be without biological activity in the infant. GnRH antagonists have the advantage of immediate inhibitory action. They have potential application in circumstances in which agonists have been employed and, in addition, can interrupt any stage of the menstrual cycle. Clinical trials to utilise their potential antifertility action have not been performed. Use of GnRH analogues for contraception in women may require combination with low dose oestrogen and progestin and it has been proposed that such development may yield important benefits in health. When combined with testosterone, GnRH antagonists may form the basis for a male contraceptive.

Progesterone secretion has been observed to be episodic in the late luteal phase of the oestrous cycle of ewes and is apparently independent of luteinizing hormone (LH). This study investigated the effects of suppressing the pulsatile release of LH in the early or late luteal phase on the episodic secretion of progesterone. Six Scottish Blackface ewes were treated i.m. with 1 mg kg-1 body weight of a potent gonadotrophin-releasing hormone (GnRH) antagonist on either day 4 or day 11 of the luteal phase. Six ewes received saline at each time and acted as controls. Serial blood samples were collected at 10 or 15 min intervals between 0 and 8 h, 24 and 32 h, and 48 and 56 h after GnRH antagonist treatment and daily from oestrus (day 0) of the treatment cycle for 22 days. Oestrous behaviour was determined using a vasectomized ram present throughout the experiment. Progesterone secretion was episodic in both the early and late luteal phase with a frequency of between 1.6 and 3.2 pulses in 8 h. The GnRH antagonist abolished the pulsatile secretion and suppressed the basal concentrations of LH for at least 3 days after treatment. This suppression of LH, in either the early or late luteal phase, did not affect the episodic release of progesterone. Daily concentrations of progesterone in plasma showed a minimal reduction on days 11 to 14 after GnRH antagonist treatment on day 4, although this was significant (P < 0.05) only on days 11 and 13. There was no effect of treatment on day 11 on daily progesterone concentration, and the timing of luteolysis and the duration of corpus luteum function was unaffected by GnRH antagonist treatment on either day 4 or day 11. These results indicate that the episodic secretion of progesterone during the luteal phase of the oestrous cycle in ewes is independent of LH pulses and normal progesterone secretion by the corpus luteum can be maintained with minimal basal concentrations of LH.

In previous studies, the LHRH antagonist detirelix, administered to stumptailed macaques during the menstrual cycle, was only partially effective in blocking pituitary-ovarian function when given during the late follicular phase. Since the antagonist was suppressive when administered during the early luteal phase, we investigated the ability of antide, a putative long-acting LHRH antagonist, to cause inhibition of the LH surge or luteal function when administered during the late follicular phase. Six animals with regular ovulatory cycles were treated on day 10 of the follicular phase with 1mg/kg antide s.c. All animals demonstrated a continued rise in serum concentrations of estradiol which were followed by an LH surge beginning 2-5 days after antide injection and serum progesterone and inhibin secretion which indicated normal luteal function. In a second experiment, six animals were treated on day 10 of the follicular phase with 3mg/kg antide s.c. In three animals, this caused a fall in serum concentrations of estradiol and the expected LH surge and rises in progesterone and inhibin secretion indicating ovulation failed to occur. In 2 animals, the LH surge was not prevented but the consequential rise in progesterone and inhibin was attenuated. In the remaining animal the cycle appeared unaffected. Pharmacokinetics of antide revealed an initial high release rate during the first 4 days (1mg/kg) or 6 days (3mg/kg) followed by a period of sustained release at a relatively low level. These results show that antide is partially effective in blocking ovulation at a high dose in the macaque and may result in an inadequate luteal phase, presumably as a result of its extended action.

Suppression of LH and testosterone secretion in sexually active rams by the short-term administration of an LHRH antagonist results in a compensatory increase in the release of LHRH from the hypothalamus. This is inferred from the observed increase in the frequency of LH pulses in peripheral blood during the period of recovery when the pituitary regains its responsiveness to LHRH. To investigate the nature of the inhibitory feedback signal which triggers this compensatory response, a single intravenous injection of 1 mg of an LHRH antagonist (28 micrograms/kg; N-Ac-D-pCl-Phe 1, D-pCl-Phe 2, D-Trp 3, D-hArg (Et 2) 6, D-Ala 10, LHRH) was given to groups of intact, testosterone-implanted castrated and castrated rams housed under stimulatory short days. Pulsatile LH secretion was monitored in blood samples collected every 10 min for 34 h. The treatment caused an immediate blockade of LH pulses in all three groups of rams followed by a progressive recovery of LH secretion from 12-30 h. Compared to the pretreatment period, intact rams showed a significant increase in frequency of LH pulses during the recovery period. Castrated rams did not show this increase, with or without supplementary testosterone. Since the circulating testosterone concentration decreased after the blockage of LH secretion in the intact rams, but not in the castrated or testosterone-implanted castrated rams, we conclude that it is the reduction in the steroid negative feedback signal which leads to a compensatory increase in the activity of the LH pulse generator.

The use of pituitary desensitization by an LHRH agonist (buserelin) to examine pituitary-testicular function was investigated in a New World primate. Six adult male marmoset monkeys were injected s.c. with an LHRH agonist implant (1.5 mg in a rod 0.5 cm long). Pharmacokinetics, determined by radioimmunoassay of urinary buserelin, revealed a rapid initial release of the agonist followed by a steady decline during a 200-day period. The LHRH agonist treatment resulted in a rapid initial rise in plasma LH followed by a return to mean values similar to those seen in the control samples by 7 days after implantation. Using the present protocol, no evidence of subsequent pituitary desensitization or suppression of testicular function was observed, plasma concentrations of LH and testosterone remaining within the normal range during the 200-day study period. In contrast, pituitary-testicular function was suppressed in the male marmoset after blockade of pituitary LHRH receptors by an LHRH antagonist. Five adults were treated with a single s.c. injection of the antagonist [Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-Ser(Rha)6,AzGlyNH2(10)]-LHRH at a dose of 300 micrograms/kg. The LHRH antagonist induced a marked suppression of plasma LH and testosterone by 6-8 h, the low levels being maintained for 24-48 h. These results show that, whereas treatment with an LHRH antagonist can inhibit pituitary-testicular function in the male marmoset, it may be that desensitization cannot be induced by the LHRH agonist used.

The physiological roles of chicken LHRH-I and -II (cLHRH-I and -II) in the regulation of gonadotrophin release were investigated in the domestic chicken. Measurements of the neuropeptides, using specific radioimmunoassays, in brain sections cut in three planes or in grossly dissected brain areas, showed that cLHRH-II occurs in low amounts throughout the brain whereas cLHRH-I is most abundant in the diencephalon. Within the diencephalon, the largest amount of cLHRH-I occurred in the median eminence of the hypothalamus. The amount of cLHRH-I in the median eminence was higher (P less than 0.05) in laying than in out-of-lay hens. No cLHRH-II was detected in the median eminence in either reproductive state. The amount of cLHRH-I in the hypothalamus was increased (P less than 0.05) in cockerels at the onset of puberty and in somatically immature birds after castration. There were no correlated changes in the amounts of hypothalamic cLHRH-II measured in the same experimental samples. Active immunization of laying hens against cLHRH-I but not against cLHRH-II resulted in the complete regression of the reproductive system and a depression in the concentration of plasma LH. These observations, taken together, suggest that gonadotrophin secretion in the hen is more likely to be directly regulated by cLHRH-I than by cLHRH-II.

Ten endometriosis patients received luteinizing hormone releasing hormone (LH-RH) agonist (buserelin) implant injections (6.6 mg subcutaneously) at days 0, 42, 84 and 126. Serum LH and follicle-stimulating hormone (FSH) were lowered by day 14. Luteinizing hormone remained at basal concentrations while FSH returned to values in the low-normal range of the menstrual cycle by day 35. At the end of the luteal phase during which treatment commenced, estrone and pregnanediol declined and remained at postmenopausal or early follicular phase values until days 305 to 460. Time to first ovulation ranged from 321 to 481 days after starting treatment. After the initial menstruation, only three instances of bleeding occurred during treatment. Pelvic pain was relieved or markedly reduced by day 42 and remained absent throughout the period of ovarian suppression. These results indicate the potential of a long-acting LH-RH agonist implant to form the basis for the treatment of symptomatic endometriosis.

To compare the effects of transitory suppression of pituitary gonadotropin secretion by an LHRH antagonist at the mid or late follicular phase of the menstrual cycle, adult macaques with normal menstrual cycles were treated with an LHRH antagonist (N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 ]LHRH (detirelix) administered subcutaneously at a dose of 300 micrograms/kg, daily for 3 days beginning either during the mid or late follicular phase. In all eight animals treated during the mid follicular phase, serum concentrations of LH and FSH declined and remained suppressed for 4 days. This caused a fall in serum concentrations of estradiol and the expected ovulation failed to occur. During the recovery period a marked rise in serum FSH occurred followed by normal follicular development and ovulation 14.8 +/- 0.6 days after the last injection of antagonist. Of the 9 macaques given the same treatment during the late follicular phase, only in two was the expected rise in serum progesterone prevented. In 4 of the animals a transitory suppression in LH and estradiol was observed but this was followed by a recovery and occurrence of an LH surge and rise in serum progesterone indicating ovulation during the course of treatment. In the remaining 3 macaques treatment commenced on the day of the initiation of the LH surge and was associated with a progesterone rise of normal duration but lower than normal magnitude during the early luteal phase. These results show that LHRH antagonist treatment causes rapid inhibition of pituitary-ovarian function when administered up to the mid follicular phase of the cycle and is effective in blocking ovulation. The suppressive effects of the antagonist are reduced when administered during the late follicular phase. This may be due to decreased dependence of the pituitary gonadotrope on LHRH at this time and on decreased dependence of the dominant follicle on the gonadotropins.

The role of the pituitary gonadotrophins in controlling luteal function in the stumptailed macaque has been investigated by examining profiles of serum concentrations of LH, FSH, progesterone and oestradiol in daily blood samples from 13 monkeys during the menstrual cycle, and in blood samples taken at hourly intervals between 09.00 and 21.00 h on different days of the luteal phase in 13 cycles. The effects of acute withdrawal of gonadotrophins was investigated by administering a single injection of 300 micrograms LHRH antagonist/kg body weight at different stages of the luteal phase during 28 cycles. Although there were high basal values and marked fluctuations of bioactive LH during the first 4 days after the LH peak, progesterone profiles showed no corresponding short-term changes, there being a slow and steady rise in progesterone concentrations during the sampling periods. After day 5, basal LH secretion decreased, but high amplitude LH pulses were identified which were associated with episodes of progesterone secretion. Administration of the LHRH antagonist caused a suppression of bioactive LH and progesterone concentrations at all stages of the luteal phase, although some basal secretion of progesterone was maintained through the 24-h period of effective antagonist gonadotroph blockade. Luteal function recovered apparently normally in all monkeys treated in the early-mid-luteal phase. Serum concentrations of FSH and oestradiol fluctuated comparatively less during the 12-h sampling periods, and the antagonist had less suppressive effects on the concentrations of these hormones. The LHRH antagonist had no apparent effect on prolactin release.(ABSTRACT TRUNCATED AT 250 WORDS)

Incubation of preovulatory rat follicles isolated from PMSG-treated immature rats for periods of less than 24 h in the presence of LHRH or LHRH agonist resulted in stimulation of basal androstenedione and progesterone release. The stimulatory effects seen were time-dependent, occurring from 2-3 h for androstenedione and from 8-20 h incubation for progesterone. Dose-dependent stimulation occurred with both LHRH agonist (in the range 5 X 10(-10) to 10(-8) M) and native LHRH (from 10(-9) to 10(-6) M), both being in the range of the reported Ka for ovarian LHRH receptors. LHRH did not enhance the stimulatory effect of 1-100 mIU hCG on androstenedione and progesterone release. These results show for the first time that LHRH and LHRH agonist can exert a stimulatory action on androgen release from isolated preovulatory follicles. This suggests that LHRH may be acting either directly at the thecal cell level or indirectly via decreased granulosa cell aromatization.

Because of the widespread long-term clinical use of luteinizing hormone-releasing hormone (LRH) agonists, the possibility of antibody development to one of these, (D-Ser-(Butt)6) LRH (1-9) nonapeptide-ethylamide (buserelin), has been studied by examining sera from experimental animals and humans receiving long-term agonist-treatment for ability to bind 125I-labelled buserelin. None of the sera from monkeys, rabbits, dogs or rats receiving buserelin by sc injection or via minipump or from 81 patients receiving the agonist by sc injection or nasal spray had detectable antibodies. The immunochemistry of buserelin was investigated by actively immunizing male rabbits against the agonist. This also enabled us to determine the possibility of this stimulus to the immune response resulting in the generation of antibodies which could cross-react with endogenous LRH and neutralize its biological effect. All 41 rabbits immunized against unconjugated buserelin and all 23 immunized against buserelin conjugated to a protein carrier developed antibodies, those in the latter group producing highest titres. Serum from one of the animals immunized against unconjugated buserelin and from 9 of the animals immunized against conjugated buserelin had the capacity to bind 125I-labelled LRH. These LRH antibody titres were very low and were insufficient to cause suppression of serum testosterone concentrations or affect testicular weight except in one rabbit immunized against unconjugated agonist. The LRH antibody titre in this rabbit was always less than 1:100 which our other studies have indicated to be too low to produce a biological effect. It may be that the association of low LRH antibody titre with decreased testes weight in this animal was fortuitous. We conclude that the possibility of circulating antibodies being produced to buserelin or their interfering with the action of endogenous LRH is extremely low.

About one-third of human breast cancers require hormones for their continued growth and endocrine ablation or anti-hormone therapy can cause regression of these tumours. As a consequence, ovariectomy in premenopausal women or administration of an anti-oestrogen (tamoxifen) in postmenopausal women represent major options for treatment of metastatic breast cancer. Alternatively, chronic administration of agonistic analogues of luteinizing hormone-releasing hormone (LHRH) causes regression of mammary tumours in experimental animals, and such treatment has shown promise in a small series of premenopausal women with advanced breast cancer. It has been assumed that these results were achieved by suppressing the pituitary-ovarian axis, as the treatment causes a reduction in circulating levels of gonadal steroids similar to that produced by castration. However, LHRH agonists can exert major effects on tissues other than the pituitary in animals and in the human. Such findings, coupled with reports of LHRH in human breast milk and immunohistochemical evidence for the presence of LHRH-like activity in some human breast tumours, prompted us to test whether LHRH agonists could have direct antitumour effects. We now report major direct effects of LHRH and its agonists on the growth of breast tumour cells in culture.

Rats were immunized against luteinizing hormone releasing hormone (LH-RH) and ovulation and follicular development were studied 12, 24 and 48 weeks later. The abolition of regular cyclic patterns of vaginal smears and the absence of luteal tissue in all but one of 32 rats showed that the immunization was effective in blocking ovulation. Follicular growth varied between rats and appeared to be dependent on whether the inhibition of LH-RH had been sufficient to affect the secretion of basal levels of gonadotrophins. Low levels of gonadotrophins were associated with poor follicular development, uterine atrophy and leucocytic vaginal smears, whereas levels of gonadotrophins similar to those in the dioestrous controls led to adequate follicular growth in the absence of ovulation, the production of cystic follicles, uterine stimulation and persistent vaginal oestrus. A group of rats was ovariectomized 12 weeks after immunization against LH-RH; animals with low antibody titres and large follicles responded with increases in the levels of LH and FSH in the blood, whereas in those with high antibody titres and little follicular development the concentrations of gonadotrophins remained low. The reproductive capacity of rats immunized against LH-RH was tested by caging them with normal male rats from 3 weeks after immunization. Although mating occurred in three rats during the first month, no offspring were produced. No matings occurred in the remaining 41 weeks.

It is established that the blockade of the pituitary LHRH receptor by an LHRH antagonist will suppress pituitary LH secretion and reduce serum concentrations of gonadal steroids. Little is known, however, about the activity of the LHRH/LH pulse generator during this inhibitory period or during the recovery phase. To investigate this, a potent LHRH antagonist [N-Ac-D-pCl-Phe1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6, D-Ala10 LHRH was injected iv into sexually active rams and the changes in the blood plasma concentrations of LH, FSH, testosterone, and PRL were measured in samples collected every 15 min for 24-48 h. The treatment induced an immediate blockade of pulsatile LH secretion and a parallel decline in blood levels of testosterone. Plasma levels of FSH were not suppressed by treatment with the LHRH antagonist and there was no consistent effect on plasma levels of PRL. The duration of the inhibition of LH was dose dependent lasting 4.3 +/- 0.4 h, 18.0 +/- 1.0 h, and 31.8 +/- 1.3 h for the low (6 micrograms/kg), medium (36 micrograms/kg), and high (365 micrograms/kg) doses of LHRH antagonist, respectively. During the recovery period there was an approximate 2-fold increase in the frequency of LH pulses. These results suggest a compensatory response to the decline in the negative feedback effect of testosterone secretion. Even the lowest dose of antagonist elicited a decrease in the level of testosterone and an increase in LH pulse frequency. At this dose, the decline in testosterone was very transitory indicating an acute sensitivity of the hypothalamus to changes in the negative feedback signal. These results suggest that the suppression of LH and testosterone secretion in the ram by LHRH antagonist is associated with a compensatory increase in the activity of the LHRH pulse generator.

The primary function of the testosterone secreted by Leydig cells is the maintenance of spermatogenesis and hence fertility. This action of testosterone is mediated by the Sertoli cells which nourish and support the developing spermatozoa. As normal Sertoli cell function is so critically dependent on normal Leydig cell function, a regulatory influence of the Sertoli cells on the Leydig cells has been suggested. Indeed, follicle-stimulating hormone (FSH), which acts only on Sertoli cells, can also cause profound changes in Leydig cell function, although how this is effected is unknown. We recently hypothesized that the Sertoli cell might be the source of a luteinizing hormone releasing hormone (LHRH)-like factor which we detected in the interstitial fluid surrounding the Leydig cells. As injected LHRH agonists cause impairment of gonadal function and directly inhibit FSH-induced changes in Leydig cell function through specific membrane receptors, this 'LHRH-like' factor has all the correct credentials for the postulated messenger between the Sertoli and Leydig cells. Here, we strengthen this case by demonstrating that seminiferous tubules from both the rat and the stumptailed macaque (Macaca arctoides) contain a factor which has LHRH-like receptor-binding and biological activity in vitro, but which is immunologically distinct from native LHRH. We have also shown that this factor is secreted in vitro by cultured rat Sertoli cells.

Gonadotropin secretion of adult male rats was inhibited by one-week treatment with a GnRH antagonist analogue (N-acetyl-Ala1,D-p-Cl-Phe2,D-Trp3,6-GnRH, Ant.) or a GnRH antiserum (A/S). Since Ant. but not A/S binds to GnRH receptors (R), it was expected that comparison of the two treatment regimes would elucidate a physiological role for testicular GnRH-R. Furthermore, the effect of combined gonadotropin and prolactin deficiency was studied in rats treated with Ant. and bromocriptine (BR). Available pituitary GnRH-R were decreased by both Ant. and A/S (P less than 0.01), but not by BR. Testicular content of free GnRH-R was decreased by Ant. by 70-80% (P less than 0.01), probably owing to occupancy, but A/S and BR had no effect on these binding sites. Serum LH and FSH fell by about 75% with Ant. and A/S treatments (P less than 0.01), and Prl by 90% with BR (P less than 0.01). Intratesticular testosterone (T) decreased significantly with Ant. and A/S treatment (P less than 0.01-0.05), but was unaffected by BR. Only Ant. and BR treatments together, but neither alone, were able to suppress Leydig cell capacity to produce T in vitro. Relative blockade of C21 steroid side-chain cleavage by Ant. and A/S was suggested by elevated progesterone (P) and/or P/T ratios in the serum and testis tissues. Ant. and A/S had no effect on testicular LH and FSH-R, but both decreased testicular Prl-R by about 50% (P less than 0.01-0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoneutralization of LH releasing hormone (LHRH) by the i.v. administration of LHRH antiserum in the preovulatory period in the ewe resulted in a prompt abolition of the pulsatile secretion of LH without alteration of basal plasma levels of LH. This resulted in an immediate cessation of the pulsatile secretion of ovarian oestradiol, with no change in the secretion rate of androstenedione or plasma levels of FSH. The ovarian secretion rate of testosterone increased significantly. Twenty to 22h after the injection of LHRH antiserum, pulsatile secretion of LH and ovarian oestradiol and androgen secretion remained suppressed while plasma levels of FSH had increased significantly. The bolus injection of ovine LH (10 micrograms, i.v.) at this time caused a significant increase in oestradiol, androstenedione and testosterone, these responses being inversely related to the titre of LHRH antiserum in the plasma at this time. These results show that (1) the secretion of oestradiol from the preovulatory sheep follicle is entirely dependent on the pulsatile secretion of LH and (2) since the secretion of ovarian androgens was not inhibited, LH may exert a short-term control over the aromatase enzyme system controlling the conversion of androgen to oestrogen in the granulosa cell of the preovulatory sheep follicle.

Homogenates of human luteal tissue bound radioiodinated luteinizing hormone releasing hormone agonist. Specific binding was both time- and temperature-dependent. Native LHRH and two LHRH agonists competed for binding, whereas TRH, somatostatin and oxytocin did not, indicating that the binding sites were specific. The apparent Ka values were 2 X 10(7)M-1 for both LHRH agonists and 10(6)M-1 for native LHRH. This is the first demonstration of specific binding of LHRH to human ovarian tissue. No binding could be detected to ovarian tissue from postmenopausal women.

Daily treatment of immature (30-day-old) male rats for 40 days with 50 ng of an agonist of LH-RH impaired normal development of Leydig cell function. This treatment partly or completely inhibited maturational increases in (1) the serum levels of testosterone, (2) seminal vesicle weight, (3) the in-vitro steroidogenic responsiveness of the testis, and (4) the in-vitro testicular binding of 125I-labelled hCG. In contrast, twice-weekly treatment of immature or adult rats with 50 ng LH-RH agonist had only minor effects on Leydig cell function, although hCG-binding was always significantly reduced. Testicular growth in immature rats was unaffected by daily injection of LH-RH agonist whereas twice-weekly treatment caused a small reduction in weight. None of the treatments had any major consistent effect on the pituitary or serum levels of gonadotrophins and prolactin, although daily treatment with the LH-RH agonist clearly reduced the responsiveness of the pituitary to the agonist, in terms of the amounts of LH and FSH released.

It is well established that the administration of high doses of luteinizing hormone (LH) or human chorionic gonadotropin (HCG) to animals can impair gonadal function in both the male and female. These effects can be duplicated by the administration of LH releasing hormone (LHRH) or its agonists to intact animals, including man. Somewhat surprisingly, recent results have demonstrated that similar impairment of gonadal function by LHRH and its agonists can be achieved in the absence of the pituitary (for review see ref. 7), and the demonstration of specific, high-affinity binding sites for LHRH and its agonists on ovarian luteal cells and on testicular Leydig cells, could mean that LHRH-like factors are involved in the local regulation of the gonads. A further intriguing finding is the striking similarity between the direct, inhibitory effects of LHRH and LH/HCG on the gonads; for example, both treatments reduce LH receptor numbers and the steroidogenic responsiveness of rat Leydig cells. We now offer a possible explanation for this similarity by demonstrating that HCG treatment stimulates the testicular production of a biologically active LHRH-like factor, suggesting that the inhibitory actions of HCG on the Leydig cell may be mediated by the local production of this factor.

A dose of 100 microliter of a potent ovine LHRH gamma globulin inhibited ovulation in the cyclic rat when administered at 12:00 h on the day of pro-oestrus. A dose of 10 ml of the preparation was administered i.v. to female stumptailed macaques to achieve circulating antibody titres 3-4-fold higher than in the rat. In an ovariectomized macaque, this caused a marked fall in serum concentrations of LH to less than 10% of pretreatment values and also a significant, though less pronounced, fall in FSH. Six monkeys were treated with the LHRH gamma globulin during the mid-late follicular phase of the cycle. In 2 monkeys in which serum oestradiol concentrations were less than 100 pg/ml at the time of antibody administration, the rising oestradiol levels were abruptly suppressed and the normal mid-cycle LH surge failed to occur. Serum concentrations of LH and FSH declined to low levels for 8-10 days after which time normal follicular development occurred. In the remaining 4 monkeys in which follicular development was more advanced as indicated by serum oestradiol concentrations of greater than 100 pg/ml, the antibodies induced either a transient decline or had no effect on the rising serum concentration of oestradiol. An LH/FSH surge followed by a rise in serum progesterone occurred in these macaques. When the antibodies were administered to a further 6 macaques, which had also been treated with oestradiol benzoate during the early follicular phase to induce an LH surge, the neutralization of LHRH again failed to block the surge even when the dose of antibody was increased to 20 ml. The results show that LHRH antibodies were unable to block the LH surge in the macaque. They contrast with results obtained with LHRH immunoneutralization in the sheep, rat, hamster, mouse and bird and suggest that the ability of oestrogen to induce an LH surge by acting directly on the LHRH-primed anterior pituitary gland is more dominant in the primate.

The hypothalamic-pituitary-ovarian axis of female rats was disrupted at the site of LHRH stimulation by active immunization against LHRH or at the site of LH action by active immunization against LH. Active immunization against LH was associated with an increase in pituitary LHRH receptors to levels comparable to control values at pro-oestrus whereas immunization against LHRH led to a marked reduction in receptor numbers. Ovarian LHRH receptor concentrations were increased by both treatments. It is concluded, therefore, that (1) LHRH receptors in the pituitary and ovary are not concomitantly controlled, and (2) pituitary receptor numbers are primarily under positive autoregulatory control by LHRH and that ovarian LHRH receptor concentrations may be under long-term influence of LH.

Nine adult female stumptailed monkeys (Macaca arctoides) treated chronically with daily s.c. injections of D-Ser(But)6,des-Gly10,Proethylamide9, luteinizing hormone releasing hormone (LH-RH agonist) to inhibit ovulation, were tested for their ability to respond to an oestrogen provocation test (positive feedback). On two occasions during treatment with the LH-RH agonist (first test between 10 and 35 weeks after treatment had started; second test between 30 and 60 weeks after) the animals were given an i.m. injection of 50 micrograms oestradiol benzoate/kg in arachis oil. Controls were tested between days 1 and 5 of the normal cycle. In the control monkeys oestrogen induced a clear rise in LH and FSH concentrations in the blood, with highest values between 48 and 60 h after treatment. In marked contrast, none of the animals treated with LH-RH agonist showed this pattern of response. In the first test, five of the nine monkeys showed no rise in LH while in the remaining animals the response was diminished or delayed, and in the second test all none failed to show a significant rise in LH. In all agonist-treated monkeys oestrogen failed to cause an FSH rise in either test. Thus, chronic treatment with LH-RH agonist in the monkey either abolishes, diminishes or delays the oestrogen-induced LH surge. Since, in the primate, oestrogen appears to release LH by acting directly on the pituitary gland exposed to pulses of LH-RH from the hypothalamus it appears that the LH surge is inhibited primarily because the pituitary gland has been exposed to prolonged stimulation by the agonist.

With an indirect immunofluorescence technique and an antiserum raised against synthetic LHRH, immunofluorescent granules were observed in fine radially oriented fibres in the palisade layer of the zoma externa in both the anterior and the posterior divisions of the median eminence in the greenfinch. There was no specific immunofluorescence in the tractus supraoptico-hypophyseus. It is concluded that the greenfinch median eminence contains material which is similar to mammalian LHRH.

Several lines of evidence have implicated the endogenous opioid peptides in the regulation of masculine sexual behaviour. However, although the opioid related peptides alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH) have been shown to affect lordosis behaviour in the female rat, there is as yet no evidence for a role of the endogenous opiates in the regulation of female sexual behaviour. We present here evidence that the endogenous opiates in the mesencephalic central grey (MCG) are involved in the control of lordosis behaviour in the female rat.